Digital holography has been experiencing a rapid growth over the last several years, together with the availability of cheaper and better digital components as well as more robust and faster reconstruction algorithms, to provide new microscopy modalities that improve various aspects of conventional optical microscopes. In an effort to achieve wide-field on-chip microscopy, the use of unit fringe magnification (F˜1) in lens-free in-line digital holography to claim an FOV of ˜24 mm2 with a spatial resolution of <2 μm and an NA of ˜0.1-0.2 has been demonstrated. See Oh C. et al., On-chip differential interference contrast microscopy using lens-less digital holography, Opt Express.; 18(5):4717-4726 (2010) and Isikman et al., Lens-free Cell Holography On a Chip: From Holographic Cell Signatures to Microscopic Reconstruction, Proceedings of IEEE Photonics Society Annual Fall Meeting, pp. 404-405 (2009). This work used a spatially incoherent light source that is filtered by an unusually large aperture (˜50-100 μm diameter); and unlike most other lens-less in-line holography approaches, the sample plane was placed much closer to the detector chip rather than the aperture plane, i.e., z1>>z2. This unique hologram recording geometry enables the entire active area of the sensor to act as the imaging FOV of the holographic microscope since F˜1.
More recently, a lens-free super-resolution holographic microscope has been proposed which achieves sub-micron spatial resolution over a large field-of-view of e.g., ˜24 mm2. See Bishara et al., “Holographic pixel super-resolution in portable lensless on-chip microscopy using a fiber-optic array,” Lab Chip 11, 1276 (2011). The microscope works based on partially-coherent lens-free digital in-line holography using multiple light sources (e.g., light-emitting diodes—LEDs) placed at ˜3-6 cm away from the sample plane such that at a given time only a single source illuminates the objects, projecting in-line holograms of the objects onto a CMOS sensor-chip. Because the objects are placed very close to the sensor chip (e.g., ˜1-2 mm) the entire active area of the sensor becomes the imaging field-of-view, and the fringe-magnification is unit. As a result of this, these holographic diffraction signatures are unfortunately under-sampled due to the limited pixel size at the CMOS chip (e.g., ˜2-3 μm). To mitigate this pixel size limitation on spatial resolution, several lens-free holograms of the same static scene are recorded as different LEDs are turned on and off, which creates sub-pixel shifted holograms of the specimens. By using pixel super-resolution techniques, these sub-pixel shifted under-sampled holograms can be digitally put together to synthesize a smaller effective pixel size of e.g., ˜300-400 nm, which can now resolve/sample much larger portion of the higher spatial frequency oscillations within the lens-free object hologram. Unfortunately, the imaging performance of this lens-free microscopy tool is still limited by the detection SNR, which may pose certain limitations for imaging of e.g., weakly scattering phase objects that are refractive index matched to their surrounding medium such as sub-micron sized bacteria in water.
One approach to imaging small particles using lens-free holographic methods such as those disclosed above include the use of smaller pixel sizes at the sampling (i.e., detector plane). However, such a sampling related bandwidth increase only translates into better resolution if the detection SNR is maintained or improved as the pixel size of the imager chip is reduced. Therefore, the optical design of the pixel architecture (especially in CMOS imager technology) is extremely important to maintain the external quantum efficiency of each pixel over a large angular range. While reduced pixel sizes (e.g. <1 μm) and higher external quantum efficiencies can further improve the resolution of lens-free on-chip microscopy to, e.g., the sub-200 nm range in the future, other sample-preparation approaches have been attempted to improve SNR.
Wetting thin-film dynamics have been studied in chemistry and biology and attempts have been made to incorporate the same in imaging modalities. Among these prior results, a recent application of thin wetting films towards on-chip detection of bacteria provides an approach where the formation of evaporation-based wetting films was used to enhance e.g., diffraction signatures of bacteria on a chip. See e.g., C. P. Allier et al., Thin wetting film lensless imaging, Proc. SPIE 7906, 760608 (2011). PCT Publication No. WO 2013/019640 discloses a holographic microscopic method that uses wetting films to image objects. In that method a droplet is mechanically vibrated to create a thin wetting film that improves imaging performance. PCT Publication No. WO 2013/184835 discloses a method whereby the substrate is tilted to gravitationally drive a droplet to an edge of the substrate while forming a dispersed monolayer of particles having liquid lenses surrounding the particles. Other attempts have been made to form lenses around microparticles using the evaporation of water from aqueous suspensions containing a dissolved polymer. For example, Hennequin et al., Optical Detection and Sizing of Single Nanoparticles Using Continuous Wetting Films, ACS Nano, 7 (9), pp. 7601-7609 (2013) discloses such a method for the detection and sizing of 100-200 nm particles. Still further improvements are needed to image small, nano-scale particles such as viruses and the like and in particular objects smaller than 100 nm.